Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

The effect of formaldehyde containing fixatives on ribonuclease activity

Summary 1. The inactivation of crystalline ribonuclease by formaldehyde and formaldehyde containing fixatives (Serra's solution) is demonstrated.2. The rate of inactivation is shown to be dependent uponph, formaldehyde concentration, and time of action of the fixative.3. The effect of formaldehyde containing fixatives on the RNase activity in sections from fixed tissues is discussed, and the inactivation of that enzyme system in rat pancreas is demonstrated.With 2 Figures in the Text     

Evaluation of nine different fixatives

Summary An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes ( and chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10–0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol ( and chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3–8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens. It was concluded that detection sensitivity as determined with this artifical substrate may give a good idea about the immuno-histochemical performance obtained on tissue prepared with the actual fixative; but the degree of antigenic masking in the various tissue compartments has to be taken into account if the comparisons are to be meaningful.This study was supported by the Norwegian Cancer Society, The Norwegian Research Council for Science and the Humanities, and Anders Jahre's Fund. The results were in part presented at the Symposium on Immunocytochemistry of Lymphomas, Micro 82 Conference, The Royal Microscopical Society, London, July 1982 (Histochem. J. 15:655–689, 1983)     

Cytochemical reactions of human leprosy bacilli and mycobacteria: ultrastructural implications

Leprosy bacilli harvested from freshly biopsied tissue from cases of lepromatous, borderline and histoid leprosy were, in conjunction with Mycobacterium lepraemurium and representative mycobacteria, examined cytochemically with and without their pyridine-extractable acid-fastness. Unlike the mycobacteria, unextracted leprosy bacilli failed to give a positive response to the periodic acid Schiff test or to take up Sudan black B, toluidine blue O, alkaline methylene blue or safranin O. Once their acid-fastness was removed with pyridine, leprosy bacilli were stained by all of the foregoing dyes except Sudan black B, under this condition they remained gram positive. While permanent loss of acid-fastness from leprosy bacilli always resulted in a loss of acid hematein-fixing material (Smith-Dietrich-Baker tests), the reverse was not true. Mild aqueous saponification, bromination, or sequential treatment with lipase and phospholipase D resulted in a loss of acid hematein-positivity but not acid-fastness. After pyridine extraction, bromination, or aqueous saponification, true mycobacteria lost neither their acid hematein-positivity nor their acid-fastness. The acid hematein-positive material and the acid-fastness of both leprosy bacilli and mycobacteria were lost after treatment with alkaline ethanol. These cytochemical findings are discussed in the light of what is known of the ultrastructure of leprosy bacilli and mycobacteria, and of the occurrence of a dl-3, 4-dihydroxyphenylalanine oxidase in leprosy bacilli but not in mycobacteria. An effort is made to explain the rather unique cytochemical properties of leprosy bacilli. Since pyridine-extractable acid-fastness (and acid hematein-positivity) serve to distinguish human leprosy bacilli from M. lepraemurium, one or the other, or both, are suggested as bases for differentiating these two organisms in animal experiments designed to show the in vivo propagation of human leprosy bacilli.     

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

Summary The yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 M in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMN's) or mononuclear phagocytes (MN's). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMN's or MN's cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, -glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.     

Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes

The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene glycol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.     

Effects of different fixatives on beta-galactosidase activity.

beta-Galactosidase (beta-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal beta-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed beta-Gal activity.     

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections

Summary The AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoy's fluid, Bouin's fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.     

Cytochemical and autoradiographic ultrastructural study of the hairy cell

The ribosomal nature of the Hairy cell's Ribosome lamella Complex is given by cytochemical ultrastructural reactions. Using Autoradiography after tritiated uridine incubation no labelling was observed on the nucleolus and in the ribosome lamella Complex. Abnormalities in protein synthesis are thus demonstrated.     

Cytochemical and ultrastructural aspects of aquatic carnivorous plant turions

Turions, which are modified shoot apices, are vegetative, dormant overwintering organs produced by perennial aquatic plants. In this study, the turion cytochemistry and ultrastructure of Aldrovanda vesiculosa, Utricularia vulgaris and U. stygia were compared with particular emphasis placed on storage substances. These three aquatic, rootless carnivorous plant species were studied at the end of their winter dormancy. At this stage, the turions of all species had starch as their main storage material. In contrast with A. vesiculosa, Utricularia turions were rich in protein storage vacuoles, and proteins were also accumulated as crystalline inclusions in the nuclei. All examined species accumulated lipid droplets in cells of epidermal glands.     

Perichromatin fibrils and chromatin ultrastructural pattern

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.     

Cytochemical and ultrastructural studies on normal and segregated nucleoli in meristematic cells

Summary The location of the two nucleolar components, in meristematic root tip cells ofAllium cepa has been studied. The segregation of granular and fibrous components of the nucleoli was induced by adenosine-3deoxyriboside. The relationship between the ultrastructure and the stain affinities of both nucleolar regions has been determined by using different selective nucleolar stains.The pars fibrosa appears positive in the silver, lead, zinc, and methylen blue stains and shows a high ATPase activity.     

"Hyperdiploidy" in the Purkinje neuron population: chromatin status or extra-DNA? The influence of fixatives on Feulgen-DNA

The influence of different fixatives (4% formaldehyde, methanol-acetic acid, methanol-formalin-acetic acid) and hydrolysis kinetics (performed at 23 degrees C and 60 degrees C) on the chromatin availability to the Feulgen reaction of cerebellar Purkinje neurons has been examined. The results show that formaldehyde preserves or even emphasizes the different availability to histochemical reactions for the detection of DNA. The existence of great heterogeneity in the Purkinje cell population, which cannot be detected in granule cells, is once again confirmed. The fact that mean values of DNA content are generally lower in granule cells than in Purkinje cells does not allow us to exclude that at least a small percentage of the highest values might be due to an actual extra DNA synthesis to a different extent.     

Cytochemical methods of detecting the ultrastructural localization of calcium

Conditions and potentialities of electron-cytochemical techniques for calcium detection are analysed in terms of current evidence on the role of membranes in sequestration of intracellular calcium pools. In most cases these conditions did not allow to preserve a native localization of calcium because the lability of calcium pools enclosed within membranes and the action of electron microscopic fixatives on the membrane permeability to Ca2+ and Ca-precipitating agents were not taken into account. Considering these factors it is essential that both the fixator and Ca-precipitating agent could diffuse through membranes simultaneously. The modes to ensure this essential condition as well as the reasons and ways to avoid artifacts in cytochemical studies are given.     

The effect of chemical fixatives on cell walls of Bacillus subtilis.

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.